There are limited data from resource-limited settings on antiretroviral resistance mutations that develop in patients failing second-line PI ART.pol gene and interpreted using the 2014 IAS-USA mutation list and the Stanford algorithm. Logistic regression modelling was performed to identify predictors of VF.We performed a cross-sectional virological assessment of adults on second-line ART for ≥6 months between November 2006 and December 2011, followed by a prospective follow-up over 2 years of patients with virological failure (VF) at the Hospital for Tropical Diseases, Vietnam. VF was defined as HIV RNA concentrations ≥1000 copies/mL. Resistance mutations were identified by population sequencing of the Two hundred and thirty-one patients were enrolled in the study. The median age was 32 years; 81.0% were male, 95.7% were on a lopinavir/ritonavir-containing regimen and 22 (9.5%) patients had VF. Of the patients with VF, 14 (64%) carried at least one major protease mutation [median: 2 (IQR: 1–3)]; 13 (59%) had multiple protease mutations conferring intermediate- to high-level resistance to lopinavir/ritonavir. Mutations conferring cross-resistance to etravirine, rilpivirine, tipranavir and darunavir were identified in 55%, 55%, 45% and 27% of patients, respectively. Higher viral load, adherence <95% and previous indinavir use were independent predictors of VF. The 2 year outcomes of the patients maintained on lopinavir/ritonavir included: death, 7 (35%); worsening virological/immunological control, 6 (30%); and virological re-suppression, 5 (25%). Two patients were switched to raltegravir and darunavir/ritonavir with good HIV control.High-prevalence PI resistance was associated with previous indinavir exposure. Darunavir plus an integrase inhibitor and lamivudine might be a promising third-line regimen in Vietnam. There are few data regarding the prevalence of and risk factors for PI resistance developed on second-line ART in Asia. Significant uncertainty exists regarding the risk factors for PI resistance in programmatic settings, the contribution of HIV-1 subtypes to mutation development and the clinical outcomes in patients with PI resistance on long-term second-line ART. HIV-1 subtype CRF01_AE accounts for 99% of HIV infections in Vietnam,21 which is among the Asian countries with the highest numbers of HIV infections.23 Of the 90 000 people on ART, 3% are on second-line therapy.23Because of its costs, viral load monitoring of HIV is not performed routinely. Therefore, data on virological outcome and drug resistance in patients on second-line therapy are lacking. To this end, we aimed to generate data on antiretroviral resistance profiles of HIV-1 CRF01_AE-infected patients with viraemia on second-line PI therapy at the largest HIV treatment centre in Vietnam. Our objectives were: (i) to identify the risk factors for resistance development; (ii) to describe the long-term clinical outcomes of patients with resistance maintained on a failing second-line regimen; and (iii) to investigate cross-resistance to second-generation NNRTIs and PIs to inform national policy on third-line therapy.The WHO endorses ritonavir-boosted PI (PIr)-based ART as an efficacious second-line treatment after failure of NNRTI-based first-line therapy in resource-limited settings.24 In 2011, tenofovir disoproxil fumarate replaced stavudine as a preferred NRTI backbone drug. Patients in the national programme were required to attend monthly appointments for clinical and adherence evaluation. CD4 cell count was performed every 6 months. HIV load testing was performed to confirm treatment failure when the WHO's defined clinical and/or immunological failure criteria were met.26 HIV genotyping was performed to diagnose antiretroviral resistance prior to therapy switch, and results were reported to treating clinicians. Second-line therapy included nelfinavir prior to 2006 and lopinavir/ritonavir thereafter, in combination with tenofovir disoproxil fumarate and/or zidovudine plus lamivudine.28The study was conducted at the Hospital for Tropical Diseases (HTD) in Ho Chi Minh City (HCMC). The HTD is the largest centre for HIV care in southern Vietnam, providing ART for more than 5000 patients according to the national ART programme. Until the de-centralization of care in 2011–12, the HTD had been the primary provider of second-line ART for patients living in the 17 southern provinces of Vietnam. First-line therapy was administered according to national and international guidelines and at the time of the study consisted of two NRTIs (lamivudine in combination with either zidovudine or stavudine) and one NNRTI—either nevirapine or efavirenz. Indinavir was generically and locally produced  during this time and was prescribed (without ritonavir boosting) in public and private settings for patients with treatment failure or intolerance on nevirapine before efavirenz became available in 2004.This study consisted of a cross-sectional survey of adult patients (age ≥15 years) who had been on second-line ART for at least 6 months and were on active care to identify those with VF and their drug resistance development, followed by a prospective follow-up over 2 years of patients with VF. VF was defined as at least two viral loads ≥1000 copies/mL measured 1–3 months apart after intensive adherence counselling. The patients who had been on second-line therapy for <6 months at the time of study assessment, who died or who switched therapy due to drug intolerance were excluded. The study was conducted between December 2011 and June 2014.Clinical data, including demographic information, HIV risk factors, ART history, CD4 counts, HIV viral load, genotyping results at the time of therapy switch (if available), AIDS events and therapy adherence, were obtained both retrospectively and prospectively from patients' charts and from one-on-one interviews.26 Additionally, for this study, adherence was evaluated over the 6 months preceding the time of study assessment using a simple self-reported visual analogue scale (VAS).29 For analysis, sub-optimal adherence was defined as having at least one adherence score of <95% by pill count or by the VAS and/or receiving at least one qualitative adherence assessment of ‘average’ or ‘poor’ over the preceding 6 months prior.Treating clinicians routinely assessed adherence according to the national guidelines at all clinic visits; adherence was recorded either as an estimated percentage of pills taken or as a qualitative assessment of ‘good’, ‘average’ or ‘poor’, corresponding to ≥95%, 80%–94% or <80% adherence, respectively.18 The sequences were analysed using SeqScape (Applied Biosystems). Nucleotide changes were determined by comparison with the consensus sequence pNL4-3 for HIV-1 subtype B (GenBank accession number M19921). Antiretroviral resistance mutations were identified based on the 2014 IAS-USA mutation list.30 The antiretroviral resistance profile of each patient was predicted using the Stanford resistance interpretation algorithm (http://hivdb.stanford.edu). The Rega HIV-1 subtyping tool was used to determine the HIV-1 subtype of each patient sample.31At the time of enrolment, 5 mL of EDTA blood was collected for viral load measurement using the Abbott m2000rt Real Time HIV-1 assay (limit of detection of 150 copies/mL) . Antiretroviral resistance testing was performed for patients with VF using an in-house population sequencing and sequence analysis protocol as previously described, with bidirectional coverage of the complete protease gene and reverse transcriptase codons 10–300.10-transformed) HIV RNA load at therapy switch, history of indinavir use, second-line therapy delay (defined as time in months from first detection of failure of first-line ART to time of second-line therapy initiation) and an overall measure of therapy adherence (<95% versus ≥95%). The chosen covariates were either established risk factors for ART outcome36 or were based on clinicians' observations (i.e. previous indinavir use). Both univariate and multivariable analyses were performed.The following pre-defined covariates were included in the logistic regression model: CD4 cell count and . Nelfinavir was the starting PI in 10 (4.3%) patients, but it was replaced by lopinavir/ritonavir within 12 months for all patients. A total of 36 (17.1%) patients had a history of treatment with indinavir, which was frequently prescribed at 800 mg twice daily or 400 mg three times daily. Sub-optimal adherence was identified in 12.1% patients.Figure Because of cost constraints, HIV genotyping was performed only for 173 of 231 (74.9%) patients prior to therapy switch. Figure The predicted susceptibilities to the national second-line regimens containing tenofovir disoproxil fumarate, lamivudine and lopinavir/ritonavir were evaluated for the 173 patients who had genotype results using the Stanford HIV Drug Resistance algorithm. Intermediate- to high-level resistance to tenofovir disoproxil fumarate was present in 120 of 173 (69.4%), to lamivudine in 165 of 173 (95.4%) and to lopinavir/ritonavir in 2 of 173 (1.2%).The numbers of patients predicted to receive one, two and three fully active drugs were 138 of 173 (79.8%), 25 of 173 (14.5%) and 4 of 173 (2.3%), respectively.10 copies/mL (IQR: 3.92–5.01 log10 copies/mL). Five of 231 (2.2%) patients had HIV RNA concentrations between 400 and <1000 copies/mL, 4 (1.7%) patients had HIV RNA concentrations between 150 and <400 copies/mL and the remaining 200 (86.6%) patients had undetectable viral loads.The virological outcomes of the 231 patients are shown in Figure Of the 22 patients with VF, 21 (95%) were infected with HIV-1 subtype CRF01_AE; a single patient was infected with HIV-1 CRF01_AE/B recombinant. Table Figure P = 0.039; 7.8 (95% CI: 2.1–31.0), P = 0.002; and 12.8 (95% CI: 3.7–49.8), P < 0.001, respectively]. Multivariate analysis shown in Table Table ad hoc univariate and multivariate analyses of factors associated with the development of PI resistance. The three covariates identified to be independent predictors of VF were entered into the logistic regression model. Higher viral load and previous indinavir exposure remained independent predictors of PI resistance in both univariate  and multivariate  analyses. Adherence did not predict PI resistance in the univariate or multivariate analyses.We performed 3 (range: 384–942 cells/mm3). Of these five patients, two had no major PI resistance mutations and three had only one major mutation—V82A—at VF. The remaining six patients had persistent viral replication . Table 3, range: 0–177 cells/mm3); however, there were no AIDS events over the 24 months of follow-up.The clinical outcomes of the 22 patients with VF are shown in Figure 37 Ritonavir-boosted PIs are known to have a high genetic barrier to resistance.38 The minimum plasma concentrations of ritonavir-boosted PIs far exceed the levels required to inhibit WT virus replication,40 making PIs a durable class of antiretroviral drug to be used across different patient populations. A high prevalence of PI resistance has been reported in four studies, two of which studied populations from Asia, specifically, from Cambodia 15 and India .16 The other two study populations were from West Africa: Mali 37 and Nigeria .41 Except for the study from India, where indinavir/ritonavir and atazanavir/ritonavir were commonly used, the studies in these other countries used lopinavir/ritonavir for second-line therapy. Previous exposure to generically produced un-boosted indinavir and nelfinavir was implicated in the observed high prevalence of PI resistance mutations in the reports from Asia and Nigeria, although formal analyses were lacking. Our study is the first to systematically link previous PI exposure to VF and PI resistance.We report the antiretroviral resistance profiles of patients failing second-line PI-based therapy in Vietnam. The major finding was that amongst the patients experiencing VF, 64% harboured at least one major PI resistance mutation and 60% had mutations that conferred intermediate- to high-level resistance to lopinavir/ritonavir. This level of PI resistance is significantly higher than has been previously reported in either resource-rich or resource-poor settings.42 Further, the most common PI resistance mutations detected in our cohort—M46I/L, I54V, V82A and L90M—were shown to be the first mutations to be sequentially selected by indinavir therapy.43 Cheap generically made indinavir, nelfinavir and saquinavir were available in India, China and south-east Asian countries during the same time.44 This availability likely explains the higher prevalence of PI resistance reported in the studies from Cambodia and India and suggests that the scope of PI exposure and resistance in Asia might be larger than is currently appreciated. Another reason for the high level of PI resistance observed in our study as well as these other studies is the lack of viral load monitoring, which leads to late detection of VF and accumulation of PI resistance mutations. Better understanding of the extent and determinants of PI resistance in developing countries is needed.Indinavir was generically produced in Vietnam during the early 2000s. The correct dosing was 800 mg three times daily; however, because of the high rate of side effects, many Vietnamese clinicians prescribed it at 400 mg three times daily or 800 mg twice daily. A combination of high pill burden, short half-life, food restriction, high rate of side effects and inadequate dosing likely led to inadequate plasma drug concentrations and increased the risk of PI resistance in patients. Low plasma indinavir concentration has been shown to increase the risk of developing PI resistance mutations in patients experiencing early VF.41 One reason for the observed high-level etravirine cross-resistance is programmatic. The lack of virological monitoring led to prolonged periods of undetected VF in the presence of the low-genetic-barrier drugs nevirapine and efavirenz and accumulation of resistance mutations. This effect was shown by the extensive NNRTI resistance mutations in our cohort (94% of patients with ≥1 and 32% with ≥3 major NNRTI resistance mutations). NNRTI resistance mutations have been shown to persist up to 45 months after the discontinuation of NNRTI therapy. This finding is due to the low fitness costs of these mutations on viral replication, thus explaining the slow reversion of these mutant viruses to WT in the absence of drug pressure.46 The presence of ≥3 IAS-USA-defined NNRTI resistance mutations has been associated with decreased virological response to etravirine in the DUET trials.48Among the next-generation NNRTIs and PIs potentially available as third-line drugs, there was evidence of probable intermediate or high levels of cross-resistance to etravirine, rilpivirine and tipranavir in ∼50% of patients. Cross-resistance to darunavir was less frequent (27%) and was observed only at the intermediate level. These prevalences are noticeably higher than those found in studies in similar settings.49 However the most frequent NNRTI resistance mutations selected in subtype CRF01_AE virus by nevirapine and efavirenz exposure are Y181C and G190A/S, rather than K103N.51 In the DUET trials, the presence at baseline of these substitutions was associated with impaired virological response to etravirine.48 High prevalence of cross-resistance to etravirine (60%) has been reported in several studies of CRF01_AE-infected patients in Thailand for whom first-line NNRTI-based therapy was failing.54 As efficacy data of etravirine use in south-east Asia are lacking, phenotypic assays investigating the in vitro susceptibilities of these clinical isolates would be helpful. Until then, etravirine and rilpivirine should probably be avoided as third-line drugs for patients infected with subtype CRF01_AE in south-east Asia. Our data do not support the 2010 and 2013 WHO recommendations to use etravirine in a third-line ART regimen in resource-limited settings.25 The phenotypic susceptibility of tipranavir is not as well predicted as that of darunavir by most genotypic interpretation algorithms, in particular for non-B subtypes.55 However, based on our predicted cross-resistance data, darunavir/ritonavir plus a brand-new class of antiretroviral drug, such as integrase strand transfer inhibitors (INSTIs), combined with lamivudine is a reasonable third-line option for Vietnam. As the need for third-line therapy is imminent in the developing world, clinical trials evaluating cost-effective third-line treatment strategies and regimens are needed.Another reason for high etravirine cross-resistance is the inherent genetic variability of the HIV-1 subtype CRF01_AE in south-east Asia. Etravirine was designed to work against HIV containing the NNRTI signature mutation—K103N—which is highly prevalent in HIV-1 subtype B.41 However, virological re-suppression and good immune response were achieved in five patients; these patients either had no or only one major PI resistance mutation at VF detection. A strategy combining adherence intervention and close monitoring of patients failing second-line therapy before switching to third-line therapy would be cost saving yet effective in resource-poor settings.Among the 20 patients who were maintained on the failing lopinavir/ritonavir regimen, death or worse virological/immunological control occurred for 13 patients, with accumulation of resistance mutations occurring in those who remained alive at 24 months. This finding is consistent with that of a study from Nigeria showing accumulation of PI resistance mutations in patients maintained on failing second-line therapy.Our study has limitations. The study captured VF at one point in time and only in patients who were in active follow-up. The unavoidable exclusion of the 12% who had died and the 14% who had been transferred to their respective resident provinces reduces the power of our observations. Further, PI resistance might be underestimated due to the lack of data from those who had died. Nevertheless, the study site is the largest centre for second-line therapy in Vietnam. The highly uniform HIV care system along with standardized ART regimens in the national programme allow for reasonable generalizability of our findings. We did not sequence the integrase gene in this cohort, as INSTIs are not yet available in Vietnam.In conclusion, we identified a significantly higher prevalence of PI resistance in patients failing second-line therapy in Vietnam, which was associated with previous indinavir exposure. The widespread availability of generically made PIs in Asia suggests that the scope of PI resistance might be underestimated in this region. Our data emphasize the need for viral load monitoring to limit the accumulation of NRTI and NNRTI resistance mutations, thus improving second-line treatment outcome and preserving the limited third-line therapy options. Significant cross-resistance to etravirine is common in subtype CRF01_AE-infected patients failing NNRTI therapy, suggesting that etravirine should be avoided as a third-line therapy drug. Research on cost-effective strategies and timing of third-line therapy switch are now needed.Wellcome Trust.This work was supported by the None to declare.Study concept and design: T. L., V. M. Q., C. S., S. D. and J. F. Obtaining funding: T. L., S. D., J. D. and J. F. Acquisition of data: V. P. T., V. M. Q., N. T. C., N. V. V. C. and T. L. Analysis and interpretation of the data: V. P. T. and T. L. Drafting the manuscript: V. P. T. and T. L. Critical revision of the manuscript for important intellectual content: V. M. Q., N. T. C., C. S., J. F., J. D., N. V. V. C., S. D. and G. T. All authors contributed to and approved the final manuscript.


Simulations were performed using the Monte Carlo code Geant4 to determine enhancement in absorption of low- versus high-energy X-rays by Pt and to determine dose enhancement factors (DEFs) for a Pt-sensitized tumor phantom. In vitro studies were carried out using Typ-Pt and again with carboplatin due to the unexpected in vivo toxicity of Typ-Pt. Cell survival was determined using clonogenic assays. In agreement with computations and simulations, in vitro data showed up to one log unit reduction in surviving fractions (SFs) of cells treated with 1–4 µg/ml of Typ-Pt and irradiated with 160-kV versus 6-MV X-rays. DEFs showed radiosensitization in the 50–200 keV range, which fell to approximate unity at higher energies, suggesting marginal interactions at MeV energies. Cells sensitized with 1–5 or 7 µg/ml of carboplatin and then irradiated also showed a significant decrease (P < 0.05) in SFs. However, it was unlikely this was due to increased interactions. Theoretical and in vitro studies presented here demonstrated that the tumoricidal activity of low-energy X-rays was greater than that of high-energy X-rays against Pt-sensitized tumor cells. Determining whether radiosensitization is a function of increased interactions will require additional studies.The purposes of this study were (i) to investigate the differences in effects between 160-kV low-energy and 6-MV high-energy X-rays, both by computational analysis and  High-energy linear accelerators (LINACs) are in widespread use for clinical radiation therapy. Typically, 6–15-MV LINACs generate Bremsstrahlung radiation with mean energies ranging from a few MeV up to a maximum, corresponding to the peak energy of the accelerating voltage. Megavoltage (MV) radiation has been used clinically due to its much greater depth of penetration compared with low-energy X-rays in the 100–250 kV range. The therapeutic efficacy of high versus low-to-medium energy (E < 100 keV) X-rays in combination with radiosensitization by high atomic number elements, (high-Z or HZ), such as Au  and to aet al. (NO3CN)](NO3)2 was synthesized in situ according to a published procedure (NO3)2. The mixture was refluxed with stirring for 2 h, and then excess pyridine was removed by evaporation. The yellow-orange solid was washed with three portions of 1:3 (v:v) acetonitrile/ether and dried with a stream of argon . 1H NMR : δ 7.64 , 7.75 , 7.88 , 8.32 , 8.43 , 9.08 . ESI-MS m/z = 253.6 corresponds to [Pt(typ)(py)]2+. The 4-h IC50 of this compound was 3.5 μg/ml, and a concentration of 2 μg/ml resulted in 90% survival [The precursor complex [Pt(typ) for 4 h, trypsinized, spun down, re-suspended in fresh media, transferred into sterile 2-ml tubes, and then irradiated. Prior to irradiation, the cells were placed in a plastic holder, and water was added to a level of 1-cm depth, which provided back-scatter . The dose was calculated from data tables established when the LINAC was commissioned, incorporating the fall-off with distance, attenuation in media, and scatter, both internally in the LINAC and within the apparatus. Irradiations were performed at a source-to-axis distance (SAD) of 100 cm with a field-size of (25 × 25) cm at the isocenter. Tubes containing the cells were placed in an acrylic block drilled specifically for the shape of the tubes. The depth of the tubes' centers from the block edge was 2 cm. The block was setup so that the gantry faced the tubes perpendicularly, and the center of the block was placed at isocenter. Dose was calculated to the tube-center depth (2 cm). It should be noted that the acrylic isn't solid water, but given the current setup there was minimal error by assuming it was water. The 160-kV X-ray irradiations were performed using an RS2000 Small Animal Irradiator . The irradiation chamber floor was calibrated to deliver 1.13 Gy/min to that height.Megavoltage 6-MV X-irradiations were performed using a Siemens Medical Systems  LINAC located in the OSU Department of Radiation Oncology. The LINAC dose rate at the position of the cells was 2.2 Gy/min. The F98 glioma  and B16 in vitro studies were carried out with Typ-Pt. Clonogenic assays were performed by plating F98 glioma cells into Falcon Multiwell™ plates, incubating them overnight, and then exposing them to 0–4 µg/ml of Typ-Pt. After a 4-h incubation period, the cells were trypsinized, centrifuged, re-suspended in Pt-free medium and transferred to 1.5-ml microcentrifuge tubes. They then were irradiated with a 7-Gy dose of either 160-kV X-rays or 6-MV photons, as described above. Surviving fractions (SFs) were calculated by counting cell colonies composed of >50 cells under a dissecting microscope. The SFs of irradiated cells were normalized to highlight the effects of radiosensitization. A second study was performed to evaluate the effects of varying the X-ray dose. Cells were treated with a non-cytotoxic concentration of Typ-Pt (2 μg/ml) and then irradiated with either 160-kV or 6-MV X-rays at doses of 0, 1, 2.5, 5, 10 and 15 Gy. Since the purpose of performing the Typ-Pt study was to compare the relative reductions in SFs of sensitized versus unsensitized cells, the ratios of the surviving fractions (SF ratios) at each X-ray dose were computed for each treatment group. These ratios were calculated by dividing the SF of the cells that had received X-irradiation alone by that of cells irradiated following exposure to Typ-Pt, resulting in the relative enhancement in cell killing with sensitization. This approach was used to demonstrate the increasing enhancement ratio as a function of the X-ray dose in LEX sensitization compared with near constancy with HEX with very little sensitization for the latter. 160 kV and 6 MV cell-killing enhancement ratios vs unsensitized cells are directly compared, albeit with larger uncertainties than SFs for a given X-ray source. However, it should be noted that if these were computed in terms of the ratios of doses leading to SFs, then the LEX enhancement would be much smaller and in agreement with the theoretical DEFs shown in Fig. Initially, in vitro radiation sensitizer using the same methods as have been described in our studies with Typ-Pt. Cells were treated with 0–5 μg/ml of carboplatin and then irradiated with 7 Gy of 160-kV X-rays. Data from a similar experiment performed with 6-MV X-rays, which were reported previously [9 cells/ml, and the amount of sensitizer in the cell was determined based on the molecular weights of Typ-Pt (631.4 Da) and carboplatin (371.2 Da). The ratios were calculated for both the 160-kV and 6-MV irradiations groups to highlight the difference in survival of radiosensitized versus unsensitized cells after irradiation with either LEX or HEX. The survival at high dose rates was very small. Therefore the ratio of the SFs of sensitized versus unsensitized cells (rather than the absolute SFs) were presented to highlight this difference.Unexpectedly, Typ-Pt was found to be highly neurotoxic when administered i.c. by CED to non-tumor-bearing Fischer rats. This technique completely bypasses the BBB, maximizes delivery of the therapeutic agent to the brain and minimizes uptake by extracranial organs and blood . Therefoeviously , have beTo further support the data obtained with the F98 glioma cell line, as well as to test the applicability of this form of radiation therapy to another type of cancer, the experiments were repeated using carboplatin and the murine B16 melanoma cell line. Clonogenic assays were performed using the same procedure as that described for the F98 glioma cells, as described above. B16 cells first were exposed to varying concentrations of carboplatin (1–7 μg/ml) and then irradiated with an X-ray dose of 3 Gy to better delineate the difference in survival. For the second part of the study, the cells were incubated with 2.5 μg/ml of carboplatin for 4 h and then irradiated with varying doses of either 6-MV photons or 160-kV X-rays (0–15 Gy) to obtain an independent verification of the data obtained with Typ-Pt and F98 cells. The SF ratios for each X-ray dose were computed as described previously.All animal studies were carried out in accordance with National Research Council guidelines  and apprEach datapoint in the SF plots consisted of triplicate plates, and the means and standard deviations (SDs) were computed for the SFs. These were plotted in Figs t-test was used for the experiments with two groups involved, and analysis of variance (ANOVA) was used for the experiments with multiple groups involved. Holm's method was applied to adjust multiplicities to control the family-wise error rate at 0.05. SAS 9.3 software was used for analysis .Student's Results for the DEFs from numerical simulations using Geant4 corresponding to 160-kV and 6-MV spectral distributions and Pt homogeneously dispersed in a water-containing phantom are shown in Fig. vis-à-vis the photon scattering component. The PE absorption via photoionization was compared with the total X-ray absorption as a function of depth in the (H2O + Pt) phantoms. The total attenuation coefficients (κs) including photon scattering with the PE cross-sections of Pt and water, are shown in Fig. c occurred at ∼400 keV. Photoionization of water was negligible at these high energies. The efficiency of Pt as a radiation sensitizer rapidly decreased beyond the K-edge (78.4 keV) as the photon scattering component became increasingly dominant. The total attenuation coefficient approached a constant asymptotic value above Ec. There was no significant increase in energy deposition for radiosensitized phantoms irradiated with X-rays > Ec (∼400 kV).Whereas the LEX enhancement in DEFs described in the preceding section corresponded to the total radiation flux administered to the sample, we also independently computed the contribution to radiosensitization from the PE absorption component  in the HO + Pt phA comparison of the 160-kV LEX and the 6-MV HEX spectra is shown in Fig. Next, using results from the computer program XPHOT, the photoionization or PE component, which was responsible for Auger decays and cell killing, was isolated from the total X-ray flux. A detailed analysis of the LEX and HEX sources with 160-kV and 6-MV output spectra are shown in Fig. Using Eq. 1) and flux distributions in Fig.  and fluxP < 0.005) occurred using 160-kV compared with 6-MV X-rays. This was most probably due to increased attenuation of PE absorption by Pt atoms. The SFs of the irradiated cells were normalized to 1 to highlight the increased cytotoxic effects of X-rays when used with HZ radiosensitization. Figure P < 0.0001) in the SF ratio compared with those exposed to X-rays alone. The radiosensitized cells irradiated with 6-MV photons did not show a similar increase in SFs at any X-ray dose. The concentration of 2 μg/ml of Typ-Pt, corresponding to a theoretical uptake of 1.91 × 106 Pt atoms per cell, was weakly cytotoxic, suggesting that the increase in cell death was due to radiosensitization by Pt.Cells exposed to cytotoxic concentrations of Typ-Pt varying from non- to weakly cytotoxic showed a marked decrease in SFs after irradiation with a 7-Gy X-ray dose Fig. A. The reIn agreement with both the calculations of PE versus total absorption using XPHOT, and the Monte Carlo simulations for DEFs using Geant4, F98 cells irradiated with 160-kV X-rays had lower SFs relative to the cells treated with 6-MV photons by up to a factor of 10. The most probable reason for this increase in sensitization was that the coefficients for PE absorption by Pt were considerably higher than the total absorption by water at keV energies in the LEX range. This resulted in more ionizations and consequently enhanced production of low-energy Auger electrons that act like high-LET radiation. While HEX-MV photons will interact with a HZ radiosensitizer, the number of high-LET electrons emitted would be marginal compared with those resulting from LEX sources.P < 0.05) compared with those irradiated with 6-MV photons  after an X-ray dose of 5 Gy. This difference was not significant. For cells sensitized with 2.5 µg/ml of carboplatin (4.05 × 106 atoms of Pt per cell), the SFs showed a marked decrease relative to the non-sensitized cells (P < 0.0005). While this is qualitatively consistent with our hypothesis, our quantitative results require a more nuanced interpretation. Possible reasons for these observations are explored in the Discussion section.Similar to the results obtained with Typ-Pt-treated F98 cells, those that had been exposed to increasing concentrations of carboplatin and then irradiated with 7 Gy of 160-kV X-rays consistently showed significantly decreased SFs  for theP < 0.005) for all corresponding doses of carboplatin compared with those treated with 6-MV X-rays  SF ratio relative to unsensitized cells compared with those irradiated with 6 MV. Unlike the data obtained with Typ-Pt and F98 glioma cells, however, the SF ratio for cells irradiated with 6 MV was not close to unity. This will be explored in the Discussion.The data obtained using F98 glioma cells were also confirmed using murine B16 melanoma cells. B16 cells irradiated with 3 Gy of 160-kV X-rays consistently show significantly decreased survival P < 0.00 for all 32− anion. However, based on the clinical and gross neuropathologic findings, it was decided that, in a future in vivo study, the radiosensitizing properties of carboplatin at a non-therapeutic dose of 2.5 µg in combination with either 160-kV X-rays or 6-MV photons would be evaluated.The brains of rats that received 100 and 200 µg of Typ-Pt i.c. by CED showed cerebral edema, as evidenced by a flattening of the cortical surfaces and considerable congestion of the intracerebral penetrating vessels. The brains of rats that received 200 µg of Typ-Pt also showed intracerebral hemorrhage and necrosis along the path of the needle track Fig. A and subin vitro experiments have demonstrated that 160-kV X-ray sources were more effective compared with 6-MV photons in reducing the SFs of F98 glioma cells following their exposure to either Typ-Pt or carboplatin. These observations supported our hypothesis that cell survival was dependent on the energy of the X-rays and that radiosensitization was more effective when cells were irradiated with X-rays spanning the entire region between the L- and K-shells of the HZ sensitizer. Although the amounts of Pt that were used for these simulations were high, in reality they would have been equivalent to 1 µg or 7 µg/µl of carboplatin, and as previously reported by [Quantitative simulations and orted by , a dose c. For Pt compounds, the Ec was ∼400 keV. Even with the loss of flux due to attenuation as a function of depth, the radiation dose delivered by LEX X-rays from a 160-kV source would have been approximately two to three times greater than that from 6-MV photons. This would have more than compensated for a loss in dose due to attenuation as a function of depth. Therefore, it was possible to obtain a significant dose enhancement factor via photoionization, leading to increased Auger electron production and cell killing using lower-energy X-rays. The majority of the high-energy photons were scattered along the path to the tumor or passed through it with or without a HZ radiosensitizer, presumably damaging normal tissues in the process. In contrast, lower-energy X-rays with a broad peak distribution can target both the L and K shell electrons of the HZ sensitizer and would be more effective in delivering a tumoricidal dose and sparing the intervening normal tissues. Since the K and L shell energies of Au are only slightly higher than that of Pt, the findings presented here should be equally applicable to GNPs. However, it must be emphasized that the enhanced in vitro and in vivo tumoricidal effects of the combination of X-irradiation and a Pt sensitizer may also be due to completely different radiobiologicalmechanisms [1 and G0 [In our computational studies we have shown that PE absorption decreased with energy, while coherent scattering and the much larger incoherent Compton scattering cross-sections of high-energy photons increased, even in the presence of the radiosensitizer. The scattering cross-sections eventually were dominant beyond the critical energy, Echanisms –35. As hchanisms –21 and Bchanisms , 23, whechanisms . The for1 and G0  and can 1 and G0 . This ca1 and G0 –35. In o50 = 3.5 µg/ml) compared with carboplatin (IC50 = 0.437 µg/ml [Typ-Pt was synthesized in order to have a Pt compound that had lower intrinsic cytotoxicity for F98 cells IC37 µg/ml ). Howeve7 µg/ml .c of Pt at 400 keV. This suggested that much of the dose resulted in photon scattering instead of PE. This would explain the unchanged relative survival of sensitized F98 cells after irradiation with 6-MV photons versus cells exposed to radiation alone  in SF was observed with Typ-Pt at a non-cytotoxic dose of 2 μg/ml . This strongly suggested that it was radiosensitization by Pt that caused the decrease in cell survival rather than the intrinsic cytotoxicity of the sensitizer used in combination with LEX. Although most of the enhancement in radiosensitization from the 160-kV source occurs from X-rays below the K-edge, it follows that the corresponding cell survival due to Pt Auger decays in proximity to cell nuclei versus cytoplasm [For F98 cells treated with a minimally cytotoxic dose of carboplatin (1 μg/ml), we found a slight, although not statistically significant, difference in SFs at all X-ray doses relative to unsensitized cells Fig. B. In con/ml Fig. A. It sho. et al. . The difytoplasm  was highin vivo biological significance of reducing the SF of F98 glioma cells by one log unit. As has been previously reported by Yang, Barth et al. [We found a large increase in cell death at all X-ray doses for a carboplatin concentration of 2.5 μg/ml Figs B and 5B.h et al. , this woin vitro cytotoxicity. Although in vitro experiments demonstrated radiosensitization as expected, i.c. administration of Typ-Pt was highly neurotoxic in non-tumor bearing Fischer rats and therefore precluded further studies with this compound. Fourth, our theoretical model was supported by in vitro studies in which the SFs of F98 glioma cells treated with Typ-Pt and irradiated with 160-kV rays showed a decreasing trend with radiation dose compared with a marginal decrease in SFs of cells irradiated with 6-MV photons. The F98 data were concordant with those obtained with murine B16 melanoma cells using carboplatin as the sensitizer. Finally, theoretical simulations have shown that the enhancement of up to an order of magnitude using HZ sensitizers and lower-energy X-rays may more than compensate for the 2–3-fold loss of flux with depth, even with tumors at depths of 10 cm. However, the interdependence between the X-ray dose and the tumoricidal activity of the HZ sensitizer will be investigated in more detail in future studies.The present study has extended the findings on the radiosensitizing properties of HZ agents such as GNPs reported by other investigators , 8, 10 iThis research was supported in part by a Large Interdisciplinary Grant from the Ohio State University , Voices Against Brain Cancer, and a donation from the Kevin J. Mullin Memorial Fund for Brain Tumor Research. Funding to pay the Open Access publication charges for this article was provided by internal funds from the Departments of Pathology, Chemistry and Astronomy of The Ohio State University.


In zebrafish , the use of Tb3+ or Eu3+ doped luminescence glass or CdS-QD coated glass lenses provide additional visible light to the rod and cone photoreceptor cells, and thereby improve the visual system functions. The data provide proof-of-concept for the future development of optic devices for improvement of visual system functions in patients who suffer from photoreceptor cell degeneration or related retinal diseases.We developed new optic devices – singly-doped luminescence glasses and nanoparticle-coated lenses that convert UV light to visible light – for improvement of visual system functions. Tb In humans, retinal photoreceptor cells do not regenerate after apoptosis nor do they self-repair in response to damage 1245One of the strategies for recovering the lost visual system functions in degenerating retinas is to increase the absorption of light by the remaining photoreceptor cells. In this research, we tested a hypothesis that the increase of light absorption  may improve the visual system functions in control and eye-injured animals. Using singly-doped luminescence glasses and nanoparticle-coated lenses, we converted UV light to mid- and long-wavelength green or red light or wide-spectrum white light. We tested the effect of converted light in visual performance in zebrafish  using different types of behavioral assays. The data provide direct evidence for the usefulness of converted light (from UV light) for visual improvement in vertebrate animals.3+ or Eu3+ singly-doped borate glasses and CdS-quantum dot (CdS-QD) nanoparticle-coated lenses that convert UV light to visible light which can be absorbed by human photoreceptor cells (human photoreceptor cells do not absorb UV light emitted from sunlight). Tb3+ or Eu3+ singly-doped borate glasses or CdS-QDs coated lenses are highly luminescent and transparent/translucent  on visual behaviors in control and eye-injured zebrafish models. Zebrafish retinas contain both the rod and cone photoreceptor cells10121516og units ,B. The aesponses ,B. This 3+ doped glass-converted red light and white light. Solo application of UV light or luminescence glass-converted green or red light also triggered the OKR  can be recorded the OKR , likely  the OKR . The add the OKR . The OKR glasses . This isnight blindness d mutants, light-induced photoreceptor cell degeneration models)22To investigate if the converted light may have a role in improving the visual system functions in animals with eye injuries, we measured the light threshold required to trigger visually-mediated escape responses3+ or Eu3+ doped glass-converted narrow-band green or red light produced no obvious effect on rod or cone sensitivity, co-illumination of the fish with white light and CdS-QDs converted white light led to the improvement of both the rod and cone system functions, i.e., the light threshold required to evoke escape responses decreased to −6.5 ± 0.19 log units  and −4.89 ± 0.26 log units  , the photoreceptor cells were degenerated and their behavioral rod and cone sensitivity levels were decreased. In dark-adapted nbd and light-treated animals, the rod threshold was −4.61 ± 0.29 and −3.87 ± 0.22 log units, respectively  were measured in both dark- (rod dominant) and light- (cone dominant) adapted animals. In control animals, under white light conditions, the absolute rod and cone threshold was −5.96 ± 0.22 and −4.39 ± 0.30 log units, respectively ,B. Whileanimals) ,B. In zeectively . The add animals . In lighog units . The add animals .3+ or Eu3+ singly-doped glasses or CdS-QDs coated lenses enhances visual performance in zebrafish models. The overall structure and cellular organization of zebrafish retinas are similar to humans, and the physiological functions of zebrafish photoreceptor cells are nearly identical to human photoreceptor cells, i.e., the maximum light absorption of zebrafish and human rod cells is approximately 500 nm wavelength, and in both species the peak absorptive spectra for blue, green, and red cones are 415 nm, 480 nm, and 570 nm, respectively. Note that the zebrafish retinas also contain UV-sensitive cone cells that absorb light at 360 nm101112The data demonstrated that the converted light through TbIn adult zebrafish, under normal physiological conditions the addition of luminescence glass-converted narrow-band green or red light produced no obvious effect on rod or cone threshold. However, the addition of nanoparticle-converted wide-spectrum white light improved both the rod and cone system functions. In eye-injured animals, the addition of converted light improved the animals’ visual performance. It is likely mediated by the increase of light absorption in the remaining photoreceptor cells.3+ or Eu3+ doped borate glasses or CdS-QDs nanoparticle-coated lenses can be used as an optic devices for visual enhancement in patients who suffer from photoreceptor cell degeneration or other types of eye injuries. For example, by wearing luminescence- or nanoparticle-coated glasses, extra visible light will be produced from UV light emitted from sunlight. This will facilitate retinal functions, thereby restoring some of the lost visual system functions due to photoreceptor cell degeneration or eye injuries. The data described in this paper provide proof-of-concept for the future translational research and the development of optic devices for rod- or cone-photoreceptor cell degeneration patients using luminescence- and/or nanoparticle-related materials.Based on the results from this research, it is conceivable to propose that Tb3+ and Eu3+ singly-doped potassium barium borate glasses were made to convert UV light to narrow-band color light. They were prepared using standard melting and quenching techniques. Tb4O7 (99.99%), Eu2O3 (99.99%), and analytical reagents K2CO3, BaCO3 and H3BO3 were used as raw materials. A total of 70g samples were used according to glass molar compositions, and 10K2O-20BaO-69B2O3-1.0Tb2O3 and 10K2O-20BaO-69B2O3-1.0Eu2O3 were added to improve material homogeneity. Access boron acid (3mol) was added to compensate the volatile loss during the procedures. Using high pure alumina crucibles, the samples were melted at 1,200 oC for 4 hours in air and then cast onto a cold stainless steel plate to increase the cooling rate and suppress devitrification. After being annealed to release the residual stress inside the glass, the samples were machined and polished.TbCdS-polymer nanocomposites were made to convert UV light to white light. Major procedures included the synthesis of water dispersible CdS-QDs, transformation of CdS-QDs into chloroform, and incorporation of CdS-QDs into polystyrene along with POPOP organic dye. Initially, CdS-QDs were synthesized in water using 3-MPA as a surfactant (0.5mmol cadmium chloride was dissolved in 50 ml water followed by the addition of 100 μl 3-MPA). The pH of the solution was adjusted to 10 by NaOH. Then, 0.5mmol thiourea in 10 ml of water was added and refluxed at 90 °C. The solvent was evaporated and the precipitated materials were washed with water-acetone mixture and dispersed in 20 ml water. To transfer the highly water dispersible QDs to organic solvent, 3-MPA was replaced by HVDAC followed by addition of 20 ml CdS-QDs water solution. After 10 min of incubation, the chloroform phase was separated from the water phase using a separating funnel, and CdS-QDs were obtained by evaporating the chloroform. CdS QDs and POPOP were added to the styrene, and then luperox was added for polymerization. Nanocomposites were obtained by sintering at 70 °C for 72 hours.nbd, which displays age-related degeneration of the retina), and light-induced photoreceptor cell degeneration zebrafish models were used in this study. All the experiments involving animals were carried out in accordance with the guidelines approved by the NIH. Animal protocols were approved by the University of Notre Dame IACUC. Visually-mediated animal behaviors were examined to measure the visual sensitivity of developing zebrafish larvae and adult animals. These include the startle responses, optokinetic responses (OKRs), and escape responses. Visual sensitivity was determined by measuring the minimum light that evoked visually-mediated behaviors under white light illumination , either applied alone or in combination of UV light  or luminescence glass- or nanoparticle-converted green, red, or white light. Statistical differences in visual threshold measured before and after the use of converted light were determined by the Student t-test.Wild-type, mutant  cycles. Each Petri dish hosted only one larva. Prior to the visual test, the larvae were dark adapted for 30 minutes. The stimulus (3-second flashes) was presented to the larva from above. The intensity of the light was initially set at the dimmest level and then increased in 0.5-log unit stops until the first startle responses were observed. Startle responses were defined as abrupt movements in response to light onset or within 2 seconds after light offset. Each larva was tested 5 times. The minimum light that evoked the startle responses in at least 3 out of 5 tests was required to score a light threshold.The OKR was measured in developing larvae at 104 and 128 hpf. The larvae were immobilized in 1.5% low-melting agarose in the Petri dish. As such, the larvae could not swim around but could freely rotate their eyes. The grating contrast of the strips was set at 100% (black/white). The strips were rotated from either left to right or from right to left at 10 rpm. The strips were illuminated from above and the intensity of the light was initially set at the dimmest level and increased by removing neutral density filters (in 0.5-log unit steps) until the first OKR was observed. Prior to threshold light measurement, the larvae were dark-adapted for 30 minutes. The test lasted 30 seconds, during which time at least 5 positive OKRs (in the direction of strip movement) were required to record a threshold.The visual sensitivity of adult fish was determined by using an escape response assay. Normally, zebrafish swim slowly along the wall of the container. However, when challenged by a threatening object, i.e., a black segment rotating outside the container, the fish display a robust escape response: as soon as the black segment comes into view, the fish immediately turns and rapidly swims away. By measuring the minimum light required to evoke the escape responses, the visual sensitivity of adult zebrafish can be readily determined. Prior to the test, the fish were dark adapted for 30 min, then the minimum light required to evoke escape responses was recorded. At least 3 positive escape responses out of 5 encounters (between the fish and the rotating black segment) were required to record a threshold light.How to cite this article: Li, L. et al. Luminescence- and nanoparticle-mediated increase of light absorption by photoreceptor cells: Converting UV light to visible light. Sci. Rep.6, 20821; doi: 10.1038/srep20821 (2016).


The coronary artery calcification (CAC) is clinically considered as one of the important predictors of atherosclerosis. Several studies have confirmed that endothelin-1(ET-1) plays an important role in the process of atherosclerosis formation. The aim of this study was to investigate whether big ET-1 is associated with CAC.A total of 510 consecutively admitted patients from February 2011 to May 2012 in Fu Wai Hospital were analyzed. All patients had received coronary computed tomography angiography and then divided into two groups based on the results of coronary artery calcium score (CACS). The clinical characteristics including traditional and calcification-related risk factors were collected and plasma big ET-1 level was measured by ELISA. Patients with CAC had significantly elevated big ET-1 level compared with those without CAC . In the multivariate analysis, big ET-1  appeared as an independent predictive factor of the presence of CAC. There was a positive correlation of the big ET-1 level with CACS . The 10-year Framingham risk (%) was higher in the group with CACS>0 and the highest tertile of big ET-1 (P<0.01). The area under the receiver operating characteristic curve for the big ET-1 level in predicting CAC was 0.83 , with a sensitivity of 70.6% and specificity of 87.7%.The data firstly demonstrated that the plasma big ET-1 level was a valuable independent predictor for CAC in our study. Coronary artery calcification (CAC) has long been known as an important part of atherosclerotic process. Previous autopsy investigations have found a significant association between the presence of CAC and atherosclerosis burden . Now we Endothelin-1 (ET-1) is a pleiotropic molecule best known for its action as the most potent vasoconstrictor currently identified . PreviouBig ET-1, the precursor of ET-1, is a peptide of 38 amino acids, which is cleaved by ET converting enzyme-1 (ECE-1). It has been reported that plasma big ET-1 has longer half-life and easier to be detected. Moreover, emerging evidence suggested that big ET-1 is a more accurate indicator of the degree of activation of the endothelial system. So it hThe aim of this study, therefore, was to determine whether plasma big ET-1 was associated with CAC in patients who had a manifestation of chest pain.From Feb 2011 through May 2012, five hundred and ten consecutive outpatients who had a manifestation of chest pain and underwent cardiac CT using a 64-slice multidetector CT scanner were included in the study. All of them were referred to our hospital for test of blood, echocardiography and elective coronary angiography. All the blood samples within 24 hours and CAC scan within 1 month were taken. And the other imageological examinations were taken in 48 hours after withdrawing of blood samples. Mean age of patients was 56 ±10 years and 350 of them were males.Coronary arterial disease (CAD) was defined as ≥50% luminal diameter stenosis of at least one major epicardial coronary artery in elective coronary angiography. Hypertension was defined as a repeated blood pressure of ≥140/90 mmHg (at least two times in different environments) or the use of antihypertensive drugs. Diabetes mellitus (DM) was defined as a fasting serum glucose level of ≥6.99 mmol/L on multiple occasions or the use of treatment with insulin or oral hypoglycemic agents. Patients with a history of heart failure or cardiomyopathies, renal dysfunction, hepatic failure, hemolytic disorders, concomitant inflammatory diseases, neoplastic diseases, thyroid disease, acute infectious/inflammatory conditions, and a history of coronary revascularization (percutaneous coronary intervention or coronary artery bypass graft surgery) were excluded from the study. Cardiovascular risk was assessed by Framingham risk score .The study complied with the Declaration of Helsinki, and was approved by the hospital ethics review board . Informed written consent was obtained from all patients included in this analysis.Venous blood samples were obtained from each patient at baseline upon admission. Each blood sample was immediately placed on ice and then centrifuged for 20 minutes at 4°C. Plasma separation was subsequently performed at −4°C, and the samples would be tested in two hours after blood taking. Total cholesterol (TC) was measured by enzymatic methods and the levels of Hemoglobin A1c (HbA1c) were measured using the Tosoh G7 Automate HPLC Analyzer . The plasma big ET-1 level was measured using a highly sensitive and specific commercial sandwich enzyme immunoassay . The levels of high sensitivity-CRP (hs-CRP) were determined using immunoturbidimetry .All other included biomarkers were analyzed by standard hematological and biochemical tests. The patients were classified into 3 tertiles according to their plasma big ET-1 level: Tertile 1 included patients with plasma big ET-1 between 0.08 to 0.23 pmol/L, Tertile 2: 0.24 to 0.52 pmol/L, Tertile 3: 0.53 to 3.70 pmol/L.Elective coronary angiography was performed for all enrolled patients using the standard Judkins technique, and the results were analyzed by at least two interventional physicians, as in our previous study. Only anAll computerized tomography scans were performed on a 64-row spiral CT scanner  with a 0.35-second rotation time with a pitch of 0.16 to 0.22, tube voltage of 120 kV, and tube current of 200 to 550 mA. Patients with heart rates >70 beats/min were given 25 mg to 50 mg of metoprolol  orally 1 h before scanning, unless they had known any contraindication for beta-blocker usage. All data sets were reconstructed using retrospective electrocardiography (ECG)-gated sequential scan during 40% to 80% RR interval. Data sets were used to a dedicated workstation . Calcium was defined as the presence of at least 3 contiguous pixels with a density 130 HU. The calcium score for each artery was the sum of calcium scores of the left main, left anterior descending, left circumflex and right coronary arteries according to the Agatston scoring algorithm .All analyses were performed using the SPSS version 19.0. software program . Continuous variables were expressed as means±standard deviation and categorical variables were expressed as frequencies with percentages. All continuous variables were checked with Kolmogorov–Smirnov normality test to show their distributions. Continuous variables with normal distributions were compared using the two-sample Student’s t-test and analysis of variance (ANOVA). Continuous variables with abnormal distributions were compared using the Kruskall wallis test. For categorical variables, the Chi-square test was used.The patients were grouped according to their plasma big ET-1 levels in three tertiles. We compared the risk factors for CAD and CAC between the big ET-1 tertiles. The relationship between big ET-1 and CACS, age, male gender, DM, hypertension, smoking status, serum creatinine, uric acid, TC, HbA1c, hs-CRP and LVEF were examined by Pearson's correlation analyses. A multivariable logistic regression analyses were performed to identify variables with a significant independent association with absence of CAC. To assess the value of the Big ET-1 as a predictor for CAC, receiver-operator characteristic (ROC) curve was used. To determine the locally appropriate cut-off point for the Big ET-1, the Youden index (sensitivity+specificity−1) was calculated and the corresponding cut-off value for the highest Youden index was considered as the optimal cut-off value. Post-hoc analysis using the Bonferroni analysis was done to evaluate the p values for each pair of comparison (in the figures).The alternative test hypothesis was built as two-sided for each statistical analysis. The tests were independent and so the experimentwise Type I error does not exceed 0.05 alpha levels. Significant univariate variables with P<0.05 were included in the multiple logistic regression analysis for odds ratios and 95% confidence intervals. A p-value of less than 0.05 was considered to be statistically significant.Baseline clinical and laboratory characteristics of patients according to big ET-1 tertiles were shown in Participants in higher big ET-1 tertiles were older, more likely to be smoking and associated with CAC, DM and hypertension. While they had elevated HbA1c, hs-CRP levels and low TC levels , age , smoking status , CAD , DM , hypertension , HbA1c  and hs-CRP  and drugs which may affect plasma big ET-1 level , the tertiles of big ET-1 were analysised in the multivariate analysis by Cox regression model . The finArea under the receivers operating characteristic (ROC) curves  suggesteTo evaluate the value of the 10-year Framingham risk using combination of big ET-1 and CAC in our study, patients were divided into three subgroups according to plasma big ET-1 levels and CAC. Group A (n = 103): CACS = 0 and the levels of big ET-1 in Tertile 1; Group C (n = 163): CACS>0 and the levels of big ET-1 in Tertile 3; Group B (n = 244): the participants who were not in Group A and in Group C. Comparison within the groups by Bonferroni post hoc analysis showed the combintion of big ET-1 and CAC was a significant predictor for the 10-year Framingham risk. The 10-year Framingham risk (%) was 6.9±6.2 in group A, 10.5±8.0 in group B and 13.0±11.7 in group C P<0.001, .The present study is the first clinical investigation to demonstrate the correlation between big ET-1 and CAC in patients without renal dysfunction. The data for the first time showed that plasma big ET-1 level was an independent risk factor for presence of CAC, and that Big ET-1 had a linear relationship with CACS. In addition, the Framingham risk scores were significantly lower in the individuals with both absence of CAC and lower levels of big ET-1 than that in the other groups which CACS>0 or had higher levels of big ET-1. Additionally, similar to previous studies, the results of our study indicated that age, sex and CAD were as independent predictive factors of the presence of CAC.Vascular calcification, which was considered as a passive degenerative condition of aging previously, is currently characterized as an active biomineralization process, more akin to bone formation . PatholoIn recent years, the evidence has tremendously suggested that the presence of CAC can give prognostic information for subsequent coronary events in individuals with or without CVD . In the Although CAC has been regarded as a predictor of CV events, it remains unclear whether CAC is a benign or ominous finding. The clinical imaging studies conducted in patients with acute coronary syndrome have shown a higher proportion of non-calcified plaque compared with calcified plaque,20. SeriET-1, a peptide of 21 amino acid residues, is one of a family of three proteins encoded by distinct genes that also includes Endothelin-2 (ET-2) and Endothelin-3 (ET-3). ET-1 is regarded as one of the most potent endogenous vasoconstrictor so far. It is tSince ET-1 and CAC were closely related to atherosclerosis respectively, the relationship between them is need of being confirmed. Although elevated ET-1 prompts endothelial injury or early atherosclerosis , it has Although there are few studies regarding the relationship between ET-1 and CAC, the positive correlation of ET-1 with the aorta or mitral valve calcification has already been confirmed. Wu SY et al. demonstrated endothelin content and mRNA were significantly increased in calcified VSMCs and calcified aorta in vivo. In a hiAlthough some studies have displayed the association of ET-1 or endothelial function with calcification, the underlying mechanisms of these relationships remain unclear. There are some proposed mechanisms. Firstly, ET-1 may cause artery calcification through its potential to promote VSMC phenotypes changed . EssalihThere are several limitations in the present study. First, most of the study patients were admitted to assess their CAD risk and had symptoms of chest pain at the time of CT scanning. Therefore, our patient population was different from the general population to implicate our finding to general population. Moreover, our study did not permit the determination of causality due to a cross-sectional feature. Moreover, a single center source of patients may also be a limitation. Finally, the results from relative small sample size suggested that a large cohort study is needed.In conclusion, the present data demonstrated that the plasma big ET-1 level was a valuable independent predictor of CAC in our study. The combination of big ET-1 and CAC is in good accordance with the Framingham risk score.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa , a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterologous expressed protein E17R from Exiguobacterium  was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-subfamily. Titration down to low pH values  indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate  that decayed (τ1 = 3.5 μs) into another red-shifted intermediate. This species decayed in a two-step process , to which the initial state of E17-PR was reformed with a kinetics of 2 ms. Proton transport capability of the HAAL protein was determined by BLM measurements. Additional blue light irradiation reduced the proton current, clearly identifying a blue light absorbing, M-like intermediate. The apparent absence of this intermediate is explained by closely matching formation and decay kinetics.The sequenced genome of the poly-extremophile  Halobacterium salinarum . Th. Thmax =inset in .2~3 . The fluorescence data is in line with the argument given in Balashov et al. (2012) [a ~7.5 for its counter-ion D100 [We followed pH-induced absorbance changes in E17R between pH 2 and pH 10 in order to identify de- or reprotation of amino acids in close proximity to the chromophore . As repo2~3 . Note th. (2012)  for the ion D100 .1 = 3.5 μs), followed by the formation of another red-shifted intermediate. This species is built in a two-step process with small amplitudes (τ2 = 84 μs and τ3 = 11 ms). A lifetime of τ4 = 82 ms corresponds to the final decay of all red-shifted intermediates and the concomitant formation of the parental 524 nm-form. These flash photolysis experiments did not reveal the presence of a deprotonated species (M-state). Such behaviour is reminiscent of the ESR that also has no noticeable transient concentration of an M-state [Flash photolysis as a time-resolved detection method for absorption changes allows following—after a short laser pulse (routinely 10 ns)—absorbance changes at a wide wavelength range on a time domain from few microseconds into the seconds range. Detection of absorbance changes, followed by fit procedures provides details on the lifetime of intermediate states formation and decay. In most cases, the global fit approach is used where all detected absorbance changes over time and wavelengths are subjected to one single fit model. This data evaluation yields lifetimes and also spectral shape of the intermediates through which the protein travels. Here, we followed the built-up and decay of photo-intermediates from time-resolved absorbance changes in the microsecond to second time range after excitation of E17R’s initial dark state by a nanosecond laser flash . The res M-state –60. It wE. coli cell suspensions [black lipid membrane (BLM) method [The protein pumping activity for the closely related ESR protein was measured in pensions . For E17) method . E17R waExiguobacterium sp. S17. This halotolerant, UV-resistant microorganism [Exiguobacterium sp. S17 indicated that it is closely related and branched together with other mesophile strains  clearly distant from psycrophilic strains clade: i.e. Exiguobacterium sibiricum 255–15 and Exiguobacterium antarticum B7 [In the course of a genomic project from extremophilic microbes from HAAL, a gene (E17R) predicted as belonging to the bacteriorhodopsin family was found in the genome of organism  was origorganism  being aborganism , 50. Neiticum B7 .Exiguobacterium genomes. Accordingly, the original environments of most isolates are shallow or surficial such as microbial mats, soil, rhizosphere, glacier ice, or hot springs , E17R shows highest homology to ESR for which recently a three-dimensional structure was reported . Both PRInspection of the sequences of E17R and ESR identify positions at which amino acid are different . Helix seven also shows a high degree of sequence similarity, except for the exchange V220 → F (E17R to ESR) and V224 → T in direct vicinity to the retinal-binding lysine.i = 3.5 μs, 84 μs, and 11 ms). The initial PR524 state is then formed with τ4 = 82 ms. At first glance the apparent absence of the M-intermediate in the transient absorbance changes at the measuring pH value of 7.4 is puzzling, as a de- and reprotonation is instrumental for a vectorial proton transport. In fact, the BLM measurements clearly demonstrate the presence of a photocurrent and blue light irradiation opens a short circuit pathway leading to reduce current. Thus, the lack of an M-intermediate at the pH-value chosen for this experiment is due to purely kinetic reasons , all further intermediates show continuously increasing life times (τments in ).Exiguobacterium sp. S17 as an important member of this complex microbial community. Further studies including knock-out strategies on E17R are in progress to link light-harvesting functionality to microbial physiology. High-throughput sequencing metagenomic projects on Socompa stromatolites are also being conducted to ultimately reveal the ecological significance of these versatile novel ion pumps in the structure and function of the microbial extremophilic communities. The studies offer a high window to study ancient phototrophic systems in homologous stromatolites of their Precambrian counterparts.In this work, we have successfully cloned and heterologously overexpressed a proteorhodopsin gene from a halotolerant, UV- and arsenic resistant strain originally isolated from a high-altitude Andean Lake . To our knowledge, this is the first report of a functional proteorhodopsin isolated from modern stromatolites. Its presence in a stromatolite and its functional, light-driven proton transport activity identifies S1 File(DOCX)Click here for additional data file.


We demonstrated that subcutaneously injected HMF suppressed: (1) LPS-induced losses in body weight; (2) LPS-induced microglial activation in the hippocampus; and (3) LPS-induced interleukin-1β mRNA expression in the hippocampus. These results suggest that HMF has the ability to reduce neuroinflammation in the brain.Citrus polymethoxylated flavones (PMFs) have recently been shown to suppress inflammation in peripheral tissues. In the present study, we investigated the effects of 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), one of the PMFs, on inflammation in the brain  Inflammation is a normal physiological response to infection/injury; however, its over-activation sometimes becomes toxic. Recent studies have shown that the pathophysiology of numerous diseases/lesions in the central nervous system (CNS) are also associated with neuroinflammatory responses, such as ischemic stroke , traumatO-tetradecanoylphorbol 13-acetate (TPA)-induced skin inflammation and tumor promotion in mice [2+ ionophore-stimulated rat basophilic leukemia cells (A23187-stimulated RBL-2H3 cells) [in vitro studies suggested that HMF may have also anti-inflammatory effects in CNS-related cells; that is, HMF has been shown to inhibit the production of NO and inducible nitric oxide synthase (iNOS) in cultured astrocytes, which are involved in supporting neurons in the CNS [in vivo study, HMF suppressed the microglia activation in the hippocampus following ischemia and protected from neuronal cell death [Among natural materials, citrus fruits are some of the most popular fruits worldwide and are known to be a rich source of bioactive compounds, including flavonoids, coumarins, limonoids and carotenoids. Particularly in flavonoids, polymethoxylated flavones (PMFs) and hydroxylated polymethoxyflavones are sure to be the focus of recent attention, because of their broad spectrum of biological activities . PMFs we in mice ; (4) 5,63 cells) ; and (5)3 cells) . Recent ll death . TherefoEight-week-old male C57BL/6 mice were purchased from Japan SLC . Mice in all groups were kept at 23 ± 1 °C and on a 12-h light/dark cycle (light on 8:00~20:00). Tap water and food were freely available during the experimental period. All animal experiments were carried out in accordance with the Declaration of Helsinki and the Guidelines for Animal Experimentation prepared by the Animal Care and Use Committee of Matsuyama University .s.c.) administered HMF to achieve 100 mg/kg/day , because this dose of HMF has been shown to be more than sufficient to exert its neuroprotective effects in ischemic brain [n = 6; and LPS group, n = 11) were treated with vehicle (DMSO/PEG300). A volume of HMF solution or vehicle was 0.125 mL per 25 g mouse. The s.c. administration of these samples was continued once a day for ten days (Day 1~Day 10).HMF was prepared from commercial orange oil  as described previously , and disic brain ,16. MiceSalmonella enterica serotype typhimurium) was purchased from Sigma-Aldrich  and dissolved with phosphate-buffered saline (PBS) at the concentration of 7.5 mg/mL. The surgery for the intrahippocampal administration of LPS was performed immediately after the injection of the samples (vehicle or HMF) on the eighth day (Day 8). After mice were immobilized in a stereotaxic device  under pentobarbital anesthesia, a linear skin incision was made over the bregma, and a 1-mm burr hole was drilled in the skull 2.3-mm posterior and 1.8-mm lateral to the bregma on both sides using a hand-held driller. Two microliters of LPS solution (the dose of LPS was 15 μg) were injected 2.0 mm below the surface of the skull using a 10-μL Hamilton syringe for each hemisphere; therefore, 30 μg/4 μL of sample were injected for both hemispheres in mice.LPS (from Two days after the LPS injection (Day 10), mice were anesthetized and transcardially perfused with ice-cold PBS. Their brains were then removed, and one of the hemispheres of each brain was used for immunohistochemistry while the other hemisphere was used for RT-PCR.Brains were postfixed as described in a previous study . The froTotal RNA from the hippocampal region of the mice was prepared by use of Isogen , composed of phenol and guanidine thiocyanate and then transcribed into cDNA, as described in a previous report . The synp < 0.05 was considered to be significant.Data for the individual groups are expressed as the means ± SEM. Data were analyzed by a one-factor ANOVA followed by Tukey’s multiple comparisons test . A value of p < 0.001 vs. CON group), and the HMF treatment (100 mg/kg/day) significantly suppressed LPS-induced body weight loss (#p < 0.05 vs. LPS group).In order to examine the effects of HMF on LPS-induced sickness responses, we assessed body weights two days after the injection of LPS before mice were sacrificed. As shown in p < 0.05); on the other hand, HMF (100 mg/kg/day) treatment significantly suppressed the LPS-induced activation of microglia (#p < 0.05).We investigated the effects of HMF on LPS-induced microglial overactivation. Microglial cells were immunostained with the IBA1 antibody. In all experimental groups, IBA1 immunoreactivity was detected in the stratum radiatum and stratum lacunosum-moleculare of the hippocampus that is near the LPS injection site A. IBA1-pp < 0.001), and HMF treatment suppressed its increase (#p < 0.05). TNF-α mRNA and iNOS mRNA expression were not increased by LPS, and the treatment with HMF did not change its level. COX-2 mRNA expression in the LPS group was higher than that of the CON group, but not significant (p = 0.052), and this increment showed the tendency to suppress by the treatment of HMF at the concentration of 100 mg/kg/day  4, and its expression in the brain has been detected in the meninges, ventricular ependyma, circumventricular organs, along the vasculature and in parenchymal microglia . A previMicroglial activation has been shown to activate more microglia, as well as a self-propelling progressive cycle of inflammation . The oveWe have not yet investigated the mechanism responsible for the anti-inflammatory effects of HMF. Nuclear factor-kappa B (NFκB) and/or mitogen-activated protein kinase  are known to be involved in the LPS-induced inflammatory response, and NBT was recently reported to suppress LPS-induced TNF-α, IL-1β and iNOS in BV2 microglia by inhibiting NFκB and phospho-MAPKs . The inhIn the present study, we also showed that the administration of HMF suppressed LPS-induced body weight loss . Previous.c. injection, as we previously showed that s.c. administration of HMF suppressed the ischemic-induced microglia activation and neuronal cell death in the hippocampus [In the present study, HMF was administered by pocampus . In ordeHMF had anti-inflammatory action in the brain and suppressed inflammation-induced body weight loss. HMF might be beneficial as a therapeutic/preventive compound for various neurological diseases in humans.


Lactobacillus helveticus, L. delbrueckii subsp. bulgaricus and L. casei strains that demonstrated strong ACE-inhibitory activity. The aim was to carry out a molecular study on the bioactive peptides released by the strains with the best ACE-inhibitory properties and by the strains demonstrating a calcium-binding effect. To the best of our knowledge, this is the first study of bioactive peptides in Bulgarian white cheese. Peptides with the strongest ACE-inhibitory activity were purified and sequenced. The strains were assessed for production of peptides with calcium-binding properties. These peptides were isolated, purified and sequenced. Two strains releasing bioactive peptides with the strongest ACE-inhibitory and calcium-binding activities were selected for development of cheese starters. The strain with the best ACE-inhibitory activity was L. helveticus A1, which releases the peptide Ala-Leu-Pro-Met as a main contributor to the ACE inhibition. The strain with the best calcium-binding activity was L. casei C3 releasing the peptide SpLSpSpSpE (fraction 15–20 of ß-casein) as a main contributor to calcium binding. After pilot production of cheeses with the developed starters, the ACE-inhibitory and calcium-binding effects were confirmed during the cheese ripening. The addition of the two selected adjunct cultures led to increased production of bioactive peptides in the cheese. In this way, it is possible to increase the functional properties of Bulgarian white brined cheese.Some lactic acid bacteria strains in milk media are capable of releasing bioactive peptides. In this study, we evaluated the angiotensin-converting enzyme (ACE)inhibitory activity of 180 lactic acid bacteria and selected several  L. helveticus proteases have been shown to have ACE-inhibitory activities.[in vitro.[L. casei, L. reuterii and L. gasseri increased apparent calcium absorption bone mineral content in growing rats.[Milk proteins are precursors of many biologically active peptides which are inactive within the sequence of the precursor proteins but can be released by proteolysis during milk fermentation. Milk protein-derived bioactive peptides may function as exogenous regulatory substances with hormone-like activity on the different intestinal and peripheral target sites of the mammalian organism. To date,tivities. ACE-inhiin vitro. Some bioin vitro. It was ring rats. It is noIn this study, we analysed a large number of lactic acid bacteria strains for their ability to inhibit ACE and for their Ca-binding capacity. The main goal of this study was to characterize the bioactive peptides released by the strains with the best ACE-inhibitory properties and calcium-binding effect. To the best of our knowledge, this is the first report on characterization of ACE-inhibitory peptides and Ca-binding peptides in Bulgarian cheeses. We studied the influence of selected strains with ACE-inhibitory and Ca-binding properties included in the starter on the release of functional peptides with such properties during cheese ripening.A is optical density in both ACE and the peptide fraction, B is optical density without the peptide fraction, and C is optical density without ACE. The concentration of an ACE inhibitor needed to inhibit 50% of ACE activity is defined as the 50% inhibitory concentration (IC50).ACE activity was determined by the method of Cushman and Cheung  modifiedL. helveticus A1 was analysed at a 25%–35% acetonitrile gradient, and the fraction from L. casei C3, at 35%–45%. The last purification step was performed by means of ion-exchange HPLC column Shimadzu SCX containing benzene–sulfonic cation exchange groups in Li-mode. The elution was performed at a gradient of pH 3.0 to 9.0 in citrate buffers. Finally, single peaks with ACE-inhibitory activities were collected from the three strains that demonstrated the highest total ACE-inhibitory effect. Before sequencing, the amino acid content of the purified ACE-inhibitory peptides was determined after hydrolysis with 4 mol/L methanesulphonic acid and derivatization with phenyl-isothiocianate. Initial step for the sequencing was the fixing of the C-end of peptides to arylamine-polyvinyl-difluoride membrane, following the manufacturer's instructions . The sequencing from the N-end of the peptides was performed according to Edman et al. [The supernatants were partially purified on reverse-phase cartridges as previously explained and subjected to centrifugal ultrafiltration with a 5000 Da membrane. TFA was added to the samples to 0.1% concentration and 1 mL was injected onto a reverse phase high pressure liquid chromatography (RP-HPLC) column Nucleosil C18. The gradient from 0% to 80% acetonitrile in 0.1% TFA was conducted for 45 min. Peptides were detected at 210 nm by means of a UV-detector (Shimadzu) and 1 mL fractions were collected. These fractions were freeze dried and evaluated for anti-ACE activity. The fractions demonstrating the highest ACE-inhibitory activity were run on high pressure liquid chromatography (HPLC) column Nucleodur Sphinx at specific gradient conditions for the respective fractions. For instance, the fraction from n et al. 2 was added to 10 mL of samples. After mixing for 1 min, the Ca2+ concentration was measured by means of a Ca2+ selective electrode  connected to an Orion 290A ion analyser. The calibration curve (mV/C2+) was built with standard Ca2+ solutions. The calcium bonded by the peptides was determined after subtraction of the measured free calcium from the total calcium.The samples were deproteinized and demineralized as described above. The purified samples were buffered with Tris-HCl, pH 8.0, to a final concentration of 50 mmol/L and 100 μL of 0.1 mol/L CaCl2PO4, pH 4.8) and eluent B (NaAcetate in 25% acetonitrile) at 40 °C and a flow rate of 1 mL/min. The gradient of eluent B was from 0% to 60% for 15 min. All fractions collected after ion-exchange purification were evaluated for their Ca-binding activity. The fractions with the best Ca-binding activity were freeze dried and subsequently the corresponding peptides were sequenced as described above.The supernatants from the 10 strains with the highest Ca-binding activity were ultrafiltrated through a 5000 Da membrane and subsequently loaded onto RP-cartridges Chromabond HLB. After washing with water, the peptides were eluted with 60% acetonitrile in 0.1% TFA. After evaporation of the eluent, the substances were reconstituted in 1 mL of 10% acetonitrile in 0.1% TFA. The solution was injected onto RP-HPLC column Hypersil peptide (Macherey-Nagel). Using a Shimadzu HPLC system, 1 mL fractions were collected. The eluents were: eluent 1 (0.1% TFA in water) and eluent 2 (80% acetonitrile in 0.1% TFA). The gradient of eluent 2 was from 2% to 60% for 45 min at 40 °C and a flow rate of 1 mL/min. The fractions with the best Ca-binding activity were fractionated additionally using a BioBasic-300 cation-exchange column. The eluents used were: eluent A . This strain possesses good technological properties, such as fast acidification, production of aroma compounds and strong proteolytic activity. L. helveticus A1 is one of the best candidates for development of cheese starters. The strain showing the second best ACE-inhibitory effect was L. casei C3. This strain was the best producer of Ca-binding peptides. Another important result from the study of the total ACE-inhibitory activity was the registration of such activity for one L. delbrueckii subsp. bulgaricus strain (L. bulgaricus J24). It is a very rear case for a L. delbrueckii subsp. bulgaricus strain to demonstrate such a fair value of ACE inhibition.In this work 180 strains of lactic acid bacteria from different sources were evaluated for their ability to produce bioactive peptides with ACE-inhibitory and Ca-binding effects. The initial screening was made according to the total ACE-inhibitory and Ca-binding activities. In One of the main tasks of this study was the purification of bioactive peptides with the best ACE-inhibitory and Ca-binding effects. L. helveticus A1, the peptide was found to contain four amino acid residues, and in the case of L. casei C3, five amino acids. Concerning the peptides with a Ca-binding effect, as expected, they were shown to be mainly phospho-peptides. Three amino acid residues could be phosphorilated: serine, threonine and tyrosine. In this study, we found only phosphoserine to be involved in the Ca-binding peptides. The main source of bioactive peptides with Ca-binding activity in the present research was β-casein and αS1-casein. These two casein fractions are subjected to proteolytic attacks during ripening of the cheeses much more than other fractions. The released peptide from the strain L. helveticus A1 demonstrating the best Ca-binding activity was QMEAESpISpSpSpEE. The possible source of this peptide is f 59-70 from αS1-casein. The strain L. casei C3 showed the best Ca-binding activity due to the peptide SpLSpSpSpE, corresponding to f 15-20 from ß-casein.The bioactive peptides with a Ca-binding activity and especially with an ACE-inhibitory activity are usually small peptides. In order to analyse the molecular structure of the bioactive peptides, the general Edman sequencing in solution is not suitable because the derivatizing reagent phenyl-isothiocianate cannot be separated by extraction from the small peptides. That is why we bonded these peptides onto the membrane through aryl-amine connection. In this case, the C-end remained connected during the sequencing steps. The sequences of the peptides with the strongest ACE-inhibitory and Ca-binding properties are given in The release of such small bioactive peptides as these with ACE-inhibitory and Ca-binding effects is in parallel with a strong proteolytic activity of the strains. The technological conditions, such as the ripening temperature and the duration of cheese maturation, are key factors in the breakdown of the casein fractions by proteolytic enzymes and for the release of a sufficient quantity of bioactive peptides. After pilot production of functional cheeses with the developed starters, the ACE-inhibitory and calcium-binding effects were confirmed during the cheese ripening. As a control, we used cheese produced with the starter b189, which has proven its excellent properties during many years of application. The addition of the two specially selected adjunct cultures into the cheese starters led to increased production of bioactive peptides in the cheese products.The ability of lactic acid bacteria to release bioactive peptides is strain specific and is dependent on the dairy-processing conditions. In this study, we found two strains with strong ACE-inhibitory and Ca-binding effects. The released peptides by the two strains were purified and sequenced. The addition of such strains into the cheese starters led to the release of bioactive peptides in cheese products. In this way it is possible to increase the functional properties of Bulgarian white brined cheese.


The current strategy for diagnosing prostate cancer (PCa) is mainly based on the serum prostate-specific antigen (PSA) test. However, PSA has low specificity and has led to numerous unnecessary biopsies. We evaluated the effectiveness of urinary metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), a long noncoding RNA, for predicting the risk of PCa before biopsy. The MALAT-1 score was tested in a discovery phase and a multi-center validation phase. The predictive power of the MALAT-1 score was evaluated by the area under receiver operating characteristic (ROC) curve (AUC) and by decision curve analysis. As an independent predictor of PCa, the MALAT-1 score was significantly higher in men with a positive biopsy than in those with a negative biopsy. The ROC analysis showed a higher AUC for the MALAT-1 score (0.670 and 0.742) vs. the total PSA (0.545 and 0.601) and percent free PSA (0.622 and 0.627) in patients with PSA values of 4.0-10 ng/ml. According to the decision curve analysis, using a probability threshold of 25%, the MALAT-1 model would prevent 30.2%-46.5% of unnecessary biopsies in PSA 4–10 ng/ml cohorts, without missing any high-grade cancers. Our results demonstrate that urine MALAT-1 is a promising biomarker for predicting prostate cancer risk. Prostate cancer (PCa) is the most commonly diagnosed cancer in men in the United States, with an estimated 238,599 new cases and 29,720 deaths in the year 2013 , resultiin vitro and in vivo. Furthermore, we showed that a circulating MALAT-1 fragment (MD-miniRNA) outperformed PSA in predicting prostate biopsy outcomes, suggesting that MALAT-1 may be a promising biomarker for diagnosing PCa.Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) has been described as a regulator of metastasis and motility, and its expression is associated with metastasis in non-small cell lung cancer. It is a noncoding RNA of more than 8,000 nt derived from chromosome 11q13 . MALAT-1In this study, we investigated the potential diagnostic efficacy of urinary MALAT-1 transcript in a retrospective discovery phase (n=218) and a prospective multicenter cohort (n=216). We also evaluated the potential of the MALAT-1 score and the MALAT-1 model for PCa diagnosis.Initially, 536 patients were included in this study, from which 29 samples were excluded for insufficient RNA extraction from the sediments. After quantitative RT-PCR analysis, another 73 patients were excluded, as their PSA Ct value was above 28 , indicatTo explore whether the MALAT-1 score could be a useful biomarker to diagnose PCa in all patients and in the PSA 4-10 ng/ml cohort , the MALAT-1 score was tested in the discovery phase and evaluated in the validation phase. Our results revealed that the MALAT-1 score was significantly higher in positive biopsy patients in both the overall Figure  and the Correlation analysis demonstrated that the MALAT-1 score did not correlate with other risk factors or the Gleason score . In addiIn the discovery phase, in univariable and multivariable logistic regression models, MALAT-1 score, age, tPSA, volume, %fPSA and DRE were independent risk factors in the overall cohort, while tPSA was excluded in the PSA grey zone cohort Tables . In univThe parameters estimated from the discovery phase were evaluated in the independent validation phase. In the univariable and multivariable logistic regression models, comparable independent risk factors were obtained in the overall and PSA grey zone cohorts. There was also a trend in the overall and PSA grey zone cohorts that the MALAT-1 score   and prevented more unnecessary biopsies than the base model (30.33% vs. 22.47%). At the same threshold, the MALAT-1 based model could spare more unnecessary biopsies (30.2% vs. 20.6%) while missing fewer cancer patients (0 vs. 3.8%).The current standard approach to diagnose PCa is prostate biopsy, which is mostly based on elevated PSA and/or abnormal DRE. Although the introduction of PSA has greatly improved early PCa detection and stage migration as well as reduced PCa mortality , PSA hasAlthough the incidence of PCa in China is reported to be 1.6/100,000 per year, its incidence has recently increased substantially, with an estimated incidence of 10/100,000 in 2010 compared with 1.71/100,000 in 1993 , 25. WitMultiple derivatives, including the ratio of the free-to-total (f/t) PSA, PSA density or PSA velocity, have been developed to improve the diagnostic accuracy of PSA. %fPSA is most widely used in clinical settings to stratify the risk of PCa for men who have total PSA levels between 4 and 10 ng/ml . In our Extensive efforts have been devoted to identify novel biomarkers to diagnose PCa more accurately and at early stages to predict tumor recurrence and progression. Numerous emerging biomarkers for PCa have been discovered and tested by clinicians recently, most notably, the urinary markers PCA3 and TMPRSS2:ERG , 30. TheHowever, several limitations to this study must be acknowledged. First, the sample size was limited in both cohorts, although the initial findings were validated in a prospective multicenter cohort. Second, we did not perform a head-to-head comparison of the MALAT-1 score and the PCA3 score in our study, due to the lack of a commercial PCA3 kit in China. Third, we did not compare the performance of the MALAT-1 score with clinical nomograms  to assess the ability of the MALAT-1 score to increase the AUC of the PCPT risk calculator for predicting PCa diagnosis on biopsy. Fourth, we did not look at the value of MALAT-1 in subgroups of patients classified by initial and repeat biopsy. Future large-scale studies are needed to confirm the efficacy of the MALAT-1 score in the diagnosis of prostate cancer.In conclusion, we have clearly demonstrated that the MALAT-1 score could serve as a noninvasive biomarker for detecting PCa, especially in the PSA 4-10 ng/ml cohort. Applying a probability threshold of 25%, the MALAT-1-based model would prevent 30.2%-46.5% of unnecessary biopsies in the PSA 4–10 ng/ml cohort, while no high-grade cancers would be missed. This study is limited by the small sample size. Further large-scale studies are needed to confirm our findings.This multicenter Chinese study was approved by the institutional review board of each participating hospital. Informed consent was obtained from all patients. Subjects were recruited between May 2012 and December 2013 at three urology centers in China . All urine samples were collected from patients scheduled for prostate biopsy because of elevated serum PSA levels (≥4 ng/ml) and/or suspicious DRE. Patients with other known tumors and those receiving medical therapy known to affect serum PSA levels and/or previous PCa therapies were excluded from the study.Our study was divided into two phases Figure : (1) DisFirst-catch urine samples were collected following an attentive DRE (three strokes per lobe) before the biopsy was performed. The urine samples were immediately cooled on ice and were processed within two hours of collection. Biopsies were performed using an end-ﬁre ultrasound transducer  and an automatic 18-gauge needle . In all subjects, a 10-12 core systematic laterally directed transrectal ultrasound (TRUS)–guided biopsy was performed.g for 15 min at 4°C and the pellets were washed twice with cold PBS (1×). The sediments were then homogenized in TRIzol reagent and were used for RNA extraction or stored at −80°C until further use.The urine samples were centrifuged at 2,500 × ® Premix Ex Taq™   with an Applied Biosystems Step One Plus according to the manufacturer's recommended cycling conditions. The gene-specific sequence information for the qRT-PCR primers is listed as follows: PSAKIT-forward primer GTCTGCGGCGGTGTTCTG, PSAKIT-reverse primer TGCCGACCCAGCAAGATC; MALAT-1 forward primer CTTCCCTAGGGGATTTCAGG, MALAT-1 reverse primer GCCCACAGGAACAAGTCCTA. Briefly, 2 μl of the cDNA solution was amplified using 10 μl of SYBR® Premix Ex Taq™  (2×) , 2 μl of primers, 0.4 μl of ROX Reference Dye (50×) and nuclease-free H2O at a final volume of 20 μl. The data were analyzed with StepOne Software version v2.1 . A melt-curve analysis was enabled at the end of the amplification. Samples with PSA Ct values of >28 [Ct(PSA)-Ct(MALAT-1)×1000. All experiments were performed in triplicate. No amplification of the signal was obtained when nuclease-free water was added instead of cDNA. The data were analyzed using StepOne Software version v2.1 .Total RNA was extracted from the urine sediments using TRIzol reagent . Then, 50 ng of total RNA was treated with DNase I  prior to cDNA synthesis and was amplified using the TransPlex Complete Whole Transcriptome Amplification Kit  according to the manufacturer's instructions. qRT-PCR was performed using SYBRU-test, Student's t-test, Pearson's chi-square test and Fisher's exact test were used for statistical comparisons of continuous and categorical variables as appropriate. Univariate and multivariate logistic regressions were used to identify independent predictors of PCa upon biopsy. Co-relationships between MALAT-1 and the clinical variables were assessed by the Spearman rank correlation test. Receiver operating characteristic (ROC) curves were constructed to discriminate among different groups of patients. The area under the ROC curve (AUC) was used to assess the predictive power. The sensitivity and specificity were calculated according to the standard formulas. Decision curve analysis was used to evaluate the clinical effects of the calculators.The Mann-Whitney All of the p values were two-sided, and p<0.05 was considered to be statistically significant. All of the statistical calculations were performed using SPSS  v.17.0 , MedCalc v.10.4.7.0  and R software v.3.1.1 .